The molecular basis of mouse adaptation and virulence by human enterovirus 71

Human enterovirus 71 (HEV71), a member of the human enterovirus A species of the family Picornaviridae, is an emerging pathogen that has recently become a serious health threat in the Asia-Pacific region. Although infection normally causes mild illness that is often undiagnosed, HEV71 has emerged as the dominant cause of large outbreaks of hand, foot and mouth disease (HFMD) in the Asia-Pacific region and can cause serious central nervous system infections leading to aseptic meningitis and encephalitis with a very high mortality. A mouse model is currently the most convenient small animal model for studies of HEV71 pathogenesis and for the primary assessment of potential vaccines against HEV71. Since most HEV71 field isolates do not naturally infect mice, a mouse-adapted strain of HEV71 is required for infection of mice. The primary aim of this project was to produce mouse virulent strains of HEV71 isolated from different outbreaks of HFMD in the Asia-Pacific Region and to elucidate the molecular basis of their mouse virulence. Infectious cDNA clones of HEV71 were initially generated and used as tools to investigate the molecular basis of HEV71 virulence and pathogenesis in mice. Two infectious cDNA clones of HEV71 clinical isolates, HEV71-C2 (genogroup C2) and HEV71-C4 (genogroup C4) were successfully constructed using a low copy-number plasmid vector and an appropriate bacterial host. Transfection of cells with cDNA clones or RNA transcripts derived from these clones produced infectious viruses. Phenotypic characterisation of the clone-derived viruses was performed and they were found to have indistinguishable cell culture growth phenotypes compared to their respective wild-type viruses. However, attempts to generate infectious cDNA clones of another clinical isolate, HEV71-B5 (genogroup B5) were unsuccessful. Our previous work has shown that prior Chinese hamster ovary (CHO) cell-adaptation of HEV71 was a necessary first step in the adaptation of HEV71 to growth in murine hosts. Consequently, we selected CHO cell-adapted strains of HEV71-B5 and HEV71-C2 by serial passage in CHO cells at high multiplicities of infection. During the course of CHO cell passage, virus growth improved significantly, with increasing virus titres and the presence of cytopathic effect observed. A study of virus growth kinetics revealed that the CHO cell-adapted strains of HEV71-B5 (CHO-B5) and HEV71-C2 (CHO-C2) grew efficiently in CHO cells with maximum titres >100-fold higher than unadapted parental virus. Both CHO-B5 and CHO-C2 harboured single amino acid mutations within the VP2 capsid protein gene. CHO-B5 has an amino acid substitution of K149→I in VP2 and CHO-C2 has an amino acid substitution of K149→M in VP2. The isolate HEV71-C4 failed to adapt to CHO cells during serial passage. Infectious cDNA clone-derived populations of HEV71-C4 containing the mutations K149→I or K149→M in VP2 were generated by site-directed mutagenesis. Both mutations resulted in the ability of the virus to replicate efficiently in CHO cells, indicating that amino acid position 149 in VP2 is critical for the adaptation of HEV71 to growth in CHO cells. A mouse-virulent strain of HEV71-B5 (MP-B5) was further selected by serial passage of CHO-B5 in newborn BALB/c mice. MP-B5 induces severe disease of high mortality in newborn mice in a dose-dependent manner. Skeletal muscle is the primary site of virus replication and results in severe myositis. MP-B5 has five additional nucleotide sequence changes, two of which are located in the 5′ UTR and the three within the open reading frame (ORF). Two of the ORF mutations resulted in deduced amino acid changes in the capsid protein VP1: S241→L and K244→E; the third ORF mutation was a synonymous C→T change at nucleotide position 6072 within the 3D polymerase gene. Infectious cDNA clone-derived mutant virus populations of HEV71-26M (genogroup B3) (CHO-26M) containing the VP1 mutations identified in MP-B5 were generated in order to determine the mutation(s) responsible for mouse virulence. Only viruses expressing the VP1 (K244→E) mutation were virulent in 5-day-old BALB/c mice, indicating that the VP1 (K244→E) change is the critical genetic determinant of mouse adaptation and virulence in this model. We also modified the capsid protein of HEV71-C4 to generate a mouse virulent strain, based on the genetic information derived from our previous genogroup B3 mouse-adapted virus (MP-26M). Infectious clone-derived mutant virus populations containing the capsid protein mutations VP1: Q145→E and Q145→G were generated by site-directed mutagenesis of a full-length clone of HEV71-C4 containing CHO cell-adaptation marker, VP2 (K149→I). Viruses expressing the VP1 (Q145→E) were virulent in 5-day-old BALB/c mice with 100% mortality rate observed. Skeletal muscle appears to be the primary site of replication of this virus with limb muscle showing severe myositis. Virus was also isolated from spleen, liver, heart and brain of infected mice. The infectious cDNA clones constructed in this study have provided a valuable technical vehicle by which to dissect HEV71 virulence determinants and can be further used to study the translation and replication processes of HEV71. The mouse model of HEV71 infection has provided a fundamental basis to study the pathogenesis of HEV71. Additionally, potential HEV71 vaccine candidates can be evaluated in this more cost effective animal model. As more information emerges regarding the molecular processes of HEV71 infection, further advances may lead to the development of effective antiviral treatments and, ultimately, a vaccine-protection strategy for prevention and control of this important human pathogen

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